# US 2013/0156801 A1 · Compositions and methods for exosome targeted expression

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- Number: US 2013/0156801 A1
- Document ID: US20130156801A1
- Status: Published application
- Kind: A1
- Application number: 13/327,244
- Filed: 2011-12-15
- Published: 2013-06-20
- Earliest claimed: 2011-12-15
- Assignees: Morehouse School of Medicine
- Inventors: Vincent Craig BOND; Michael POWELL; Ming Bo HUANG; Syed ALI
- Web page: https://patentgrep.com/patent/US20130156801A1

## CPC

- C07K 14/163: {Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx}
- A61K 9/127: Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K 38/162: {from virus}
- A61P 37/04: Immunostimulants
- C07K 14/005: from viruses
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/055: containing a signal for localisation to secretory granules (for exocytosis)
- C07K 2319/50: containing protease site
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 2740/16311: concerning HIV regulatory proteins
- C12N 2740/16322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- G01N 33/543: with an insoluble carrier for immobilising immunochemicals
- G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present application relates to methods of producing exosomes. The application also provides a method for preparing a protein composition comprising culturing an exosome-producing cell expressing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest; isolating exosomes from the exosome-producing cell culture; and purifying the protein of interest from the isolated exosomes. The application further discloses compositions that comprise exosomes containing the Nef-fusion protein, as well as methods of using the Nef-fusion protein and exosomes containing the Nef-fusion protein.

## Claims

### Claim 1

A method for preparing a protein composition, comprising:
a) culturing an exosome-producing cell expressing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest;
(b) isolating exosomes from cultured exosome-producing cell; and
(c) purifying said protein of interest from isolated exosomes by chromatography.

### Claim 2 (depends on 1)

The method of claim 1, wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:3, or a variant of said Nef fragment.

### Claim 3 (depends on 1)

The method of claim 1, wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:4 or a variant of said Nef fragment.

### Claim 4 (depends on 1)

The method of claim 1, wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:5, or a variant of said Nef fragment.

### Claim 5 (depends on 1)

The method of claim 1, wherein said Nef-derived peptide comprises a Nef fragment comprising SEQ ID NO:6 or a variant of said Nef fragment.

### Claim 6 (depends on 1)

The method of claim 1, wherein said Nef fusion protein comprises an amino acid sequence encoding a functional domain selected from the group consisting of affinity tag, protease cleavage site, targeting domain, reporter, enzyme and combination thereof.

### Claim 7 (depends on 1)

The method of claim 1, wherein said exosome-producing cell is stably transformed with a vector expressing said Nef-fusion protein.

### Claim 8 (depends on 1)

The method of claim 1, wherein said exosome-producing cell is transiently transfected with a vector expressing said Nef-fusion protein.

### Claim 9

A cell line that produces exosomes containing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest, wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.

### Claim 10

A method for delivering a protein of interest to a target cell in a mammal, comprising administering to the mammal an exosome comprising a Nef-fusion protein, wherein said Nef-fusion protein comprises a Nef-derived peptide fused to said protein of interest and wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.

### Claim 11 (depends on 10)

The method of claim 10, wherein said Nef-derived peptide comprises SEQ ID NO:3.

### Claim 12 (depends on 10)

The method of claim 10, wherein said Nef-derived peptide comprises SEQ ID NO:4.

### Claim 13 (depends on 10)

The method of claim 10, wherein said Nef-derived peptide comprises SEQ ID NO:5.

### Claim 14 (depends on 10)

The method of claim 10, wherein said Nef-derived peptide comprises SEQ ID NO:6.

### Claim 15 (depends on 10)

The method of claim 10, wherein said exosome is thither loaded with a member from the group consisting of antigen, peptide, small molecule drug, and siRNA.

### Claim 16

A method for inducing an immune response in a mammal, comprising administering to a mammal an exosome comprising a Nef-fusion protein comprising a Nef-derived peptide fused to an immunogenic protein of interest, wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.

### Claim 17 (depends on 16)

The method of claim 16, wherein said immunogenic protein comprises at least a portion of a tumor antigen.

### Claim 18 (depends on 16)

The method of claim 16, wherein said exosome is isolated from an antigen-presenting cell.

### Claim 19

A method for detecting a target molecule in a sample, comprising:
contacting a sample from a subject with a Nef-fusion protein that binds specifically to said target molecule, wherein said Nef-fusion protein comprises SEQ ID NO:3, 4, 5 or 6,
detecting a binding of said target molecule in the sample to said Nef-fusion protein, and
determining a level of said target molecule in said sample,
wherein a medical condition is indicated if a level of said target molecule is outside a reference range.

### Claim 20

A pharmaceutical composition, comprising:
an exosome comprising a Nef-fusion protein containing a Nef-derived peptide fused to a protein of interest; and
a pharmaceutically acceptable carrier.

### Claim 21 (depends on 20)

The pharmaceutical composition of claim 20, wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.

### Claim 22

A Nef-fusion protein, produced by
culturing cells that produce exosomes containing said Nef-fusion protein; isolating exosomes from said cells; and purifying said Nef-fusion protein from isolated exosomes by chromotography, wherein said Nef-fusion protein comprises a Nef-derived peptide fused to a protein of interest and wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.